Recent advances in isolation, identification, and culture of mammalian spermatogonial stem cells

Abstract

Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells (SSCs). SSCs, the only male reproductive stem cells that transmit genetic material to subsequent generations, possess an inherent self-renewal ability, which allows the maintenance of a steady stem cell pool. SSCs eventually differentiate to produce sperm. However, in an in vitro culture system, SSCs can be induced to differentiate into various types of germ cells. Rodent SSCs are well defined, and a culture system has been successfully established for them. In contrast, available information on the biomolecular markers and a culture system for livestock SSCs is limited. This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process, the biology and niche of SSCs, the isolation and culture systems of SSCs, and the biomolecular markers and identification of SSCs. This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals, enhancement of human male fertility, reproductive medicine, and protection of endangered species.

Keywords: culture; identification; isolation; male germline stem cell; spermatogonial stem cell; transplantation.

Figure 1

Composition of the SSC niche. (a) Histological cross-section of the seminiferous tubules from goat testes stained with hematoxylin and eosin. Scale bar = 25 μm. (b) Schematic diagram of the seminiferous tubules and interstitial tissue from mammalian testes. SSC: spermatogonial stem cell.

Figure 2

Immunohistochemical staining of DDX4, SOX9, and WT1 in the seminiferous tubules of adult goat testes. (a) The negative control section used non-immune rabbit serum. (b) DDX4 localized in germ cells. (c) SOX9 and (d) WT1 localized in Sertoli cells. Scale bars = 25 μm. SOX9: SRY-related high mobility group-box gene 9; WT1: Wilms tumor protein 1; DDX4: DEAD-box polypeptide-4.

Figure 3

Schematic representation of the three phases of spermatogenesis in rodents. A_s: type A_single spermatogonia; A_pr: type A_paird spermatogonia; A_al: type A_aligned spermatogonia; intermediate: intermediate spermatogonia; B: type B spermatogonia.

Figure 4

Schematic representation of isolation, enrichment, and culture methods of SSCs. The testicular tissues is digested using the enzyme. Then, MACS, differential plating, Percoll gradient, or FACS is used for the enrichment of SSCs. In two-dimensional cell culture system, SSCs are cultured on feeder cells. Soft agar and MC are the most commonly used media in the three-dimensional cell culture system. SSC: spermatogonial stem cell; MACS: magnetic-activated cell sorting; FACS: fluorescence-activated cell sorting; MC: methylcellulose.